Deagglomeration of DNA nanomedicine carriers using controlled ultrasonication.

Control over the agglomeration state of manufactured particle systems for drug and oligonucleotide intracellular delivery is paramount to ensure reproducible and scalable therapeutic efficacy. Ultrasonication is a well-established mechanism for the deagglomeration of bulk powders in dispersion. Its use in manufacturing requires strict control of the uniformity and reproducibility of the cavitation field within the sample volume to minimise within-batch and batch-to-batch variability. In this work, we demonstrate the use of a reference cavitating vessel which provides stable and reproducible cavitation fields over litre-scale volumes to assist the controlled deagglomeration of a novel non-viral particle-based plasmid delivery system. The system is the Nuvec delivery platform, comprising polyethylenimine-coated spiky silica particles with diameters of âˆ¼ 200 nm. We evaluated the use of controlled cavitation at different input powers and stages of preparation, for example before and after plasmid loading. Plasmid loading was confirmed by X-ray photoelectron spectroscopy and gel electrophoresis. The latter was also used to assess plasmid integrity and the ability of the particles to protect plasmid from potential degradation caused by the deagglomeration process. We show the utility of laser diffraction and differential centrifugal sedimentation in quantifying the efficacy of product de-agglomeration in the microscale and nanoscale size range respectively. Transmission electron microscopy was used to assess potential damages to the silica particle structure due to the sonication process.

In Vivo Measurements of the Bulk Ultrasonic Attenuation Coefficient of Breast Tissue Using a Novel Phase-Insensitive Receiver.

This study describes the first in vivo acoustic attenuation measurements of breast tissue undertaken using a novel phase-insensitive detection technique employing a differential pyroelectric sensor. The operation of the sensor is thermal in nature, with its output signal being dictated by the acoustic power integrated over its surface. The particularly novel feature of the sensor lies in its differential principle of operation, which significantly enhances its immunity to background acoustic and vibration noise. A large area variant of the sensor was used to detect ultrasonic energy generated by an array of 14 discrete 3.2-MHz plane piston transducers, transmitted through pendent breasts in water. The transduction and reception capability represent key parts of a prototype Quantitative Ultrasound Computed Tomography Test Facility developed at the National Physical Laboratory to study the efficacy of phase-insensitive ultrasound computed tomography of breast phantoms containing a range of appropriate inclusions, in particular, the measurement uncertainties associated with quantitative reconstructions of the acoustic attenuation coefficient. For this study, attenuation coefficient measurements were made using 1-D projections on 12 nominally healthy study volunteers, whose age ranged from 19 to 65 years. Averaged or bulk attenuation coefficient values were generated in the range 1.7-4.6 dBcm -1 at 3.2 MHz and have been compared with existing literature, derived from in vivo and ex vivo studies. Results are encouraging and indicate that the relatively simple technique could be applied as a robust method for assessing the properties of breast tissue, particularly the balance of fatty (adipose) and fibroglandular components.

Sonocrystallisation of ZIF-8 in water with high excess of ligand: Effects of frequency, power and sonication time.

A systematic study on the sonocrystallisation of ZIF-8 (zeolitic imidazolate framework-8) in a water-based system was investigated under different mixing speeds, ultrasound frequencies, calorimetric powers and sonication time. Regardless of the synthesis technique, pure crystals of ZIF-8 with high BET (Brunauer, Emmett and Teller) specific surface area (SSA) can be obtained in water after only 5 s. Furthermore, 5 s sonication produced even smaller crystals (~0.08 µm). The type of technique applied for producing the ZIF-8 crystals did not have any significant impact on crystallinity, purity and yield. Crystal morphology and size were affected by the use of ultrasound and mixing, obtaining nanoparticles with a more spherical shape than in silent condition (no ultrasound and mixing). However, no specific trends were observed with varying frequency, calorimetric power and mixing speed. Ultrasound and mixing may have an effect on the nucleation step, causing the fast production of nucleation centres. Furthermore, the BET SSA increased with increasing mixing speed. With ultrasound, the BET SSA is between the values obtained under silent condition and with mixing. A competition between micromixing and shockwaves has been proposed when sonication is used for ZIF-8 production. The former increases the BET SSA, while the latter could be responsible for porosity damage, causing a decrease of the surface area.

Controlled Sonication as a Route to in-situ Graphene Flake Size Control.

Ultrasonication is widely used to exfoliate two dimensional (2D) van der Waals layered materials such as graphene. Its fundamental mechanism, inertial cavitation, is poorly understood and often ignored in ultrasonication strategies resulting in low exfoliation rates, low material yields and wide flake size distributions, making the graphene dispersions produced by ultrasonication less economically viable. Here we report that few-layer graphene yields of up to 18% in three hours can be achieved by optimising inertial cavitation dose during ultrasonication. We demonstrate that inertial cavitation preferentially exfoliates larger flakes and that the graphene exfoliation rate and flake dimensions are strongly correlated with, and therefore can be controlled by, inertial cavitation dose. Furthermore, inertial cavitation is shown to preferentially exfoliate larger graphene flakes which causes the exfoliation rate to decrease as a function of sonication time. This study demonstrates that measurement and control of inertial cavitation is critical in optimising the high yield sonication-assisted aqueous liquid phase exfoliation of size-selected nanomaterials. Future development of this method should lead to the development of high volume flow cell production of 2D van der Waals layered nanomaterials.

A review on possible mechanisms of sonocrystallisation in solution.

Sonocrystallisation is the application of ultrasound to the crystallisation process. The benefits obtained by sonication have been widely studied since the beginning of the 20th century and so far it is clear that ultrasound can be a very useful tool for enhancing crystallisation and controlling the properties of the final product. Crystal size, polymorphs, purity, process repeatability and lower induction time are only some of the advantages of sonocrystallisation. Even though the effects of sonication on crystallisation are quite clear, the physical explanation of the phenomena involved is still lacking. Is the presence of cavitation necessary for the process? Or is only the bubbles surface responsible for enhancing crystallisation? Are the strong local increases in pressure and temperature induced by cavitation the main cause of all the observed effects? Or is it the strong turbulence induced in the system instead? Many questions still remain and can only be appreciated with an understanding of the complexity behind the individual processes of crystallisation and acoustic cavitation. Therefore, this review will first summarise the theories behind crystallisation and acoustic cavitation, followed by a description of all the current proposed sonocrystallisation mechanisms, and conclude with an overview on future prospects of sonocrystallisation applications.

An objective comparison of commercially-available cavitation meters.

With a number of cavitation meters on the market which claim to characterise fields in ultrasonic cleaning baths, this paper provides an objective comparison of a selection of these devices and establishes the extent to which their claims are met. The National Physical Laboratory's multi-frequency ultrasonic reference vessel provided the stable 21.06kHz field, above and below the inertial cavitation threshold, as a test bed for the sensor comparison. Measurements from these devices were evaluated in relation to the known acoustic pressure distribution in the cavitating vessel as a means of identifying the mode of operation of the sensors and to examine the particular indicator of cavitation activity which they deliver. Through the comparison with megahertz filtered acoustic signals generated by inertial cavitation, it was determined that the majority of the cavitation meters used in this study responded to acoustic pressure generated by the direct applied acoustic field and therefore tended to overestimate the occurrence of cavitation within the vessel, giving non-zero responses under conditions when there was known to be no inertial cavitation occurring with the reference vessel. This has implications for interpreting the data they provide in user applications.

Influence of acoustic cavitation on the controlled ultrasonic dispersion of carbon nanotubes.

Ultrasonication is the most widely used technique for the dispersion of a range of nanomaterials, but the intrinsic mechanism which leads to stable solutions is poorly understood with procedures quoted in the literature typically specifying only extrinsic parameters such as nominal electrical input power and exposure time. Here we present new insights into the dispersion mechanism of a representative nanomaterial, single-walled carbon nanotubes (SW-CNTs), using a novel up-scalable sonoreactor and an in situ technique for the measurement of acoustic cavitation activity during ultrasonication. We distinguish between stable cavitation, which leads to chemical modification of the surface of the CNTs, and inertial cavitation, which favors CNT exfoliation and length reduction. Efficient dispersion of CNTs in aqueous solution is found to be dominated by mechanical forces generated via inertial cavitation, which in turn depends critically on surfactant concentration. This study highlights that careful measurement and control of cavitation rather than blind application of input power is essential in the large volume production of nanomaterial dispersions with tailored properties.

Progress in developing a thermal method for measuring the output power of medical ultrasound transducers that exploits the pyroelectric effect.

Progress in developing a new measurement method for ultrasound output power is described. It is a thermal-based technique with the acoustic power generated by a transducer being absorbed within a specially developed polyurethane rubber material, whose high absorption coefficient ensures energy deposition within a few mm of the ultrasonic wave entering the material. The rate of change of temperature at the absorber surface is monitored using the pyroelectric voltage generated from electrodes disposed either side of a 60 mm diameter, 0.061 mm thick membrane of the piezoelectric polymer polyvinylidene fluoride (pvdf) bonded to the absorber. The change in the pyroelectric output voltage generated by the sensor when the transducer is switched ON and OFF is proportional to the delivered ultrasound power. The sensitivity of the device is defined as the magnitude of these switch voltages to a unit input stimulus of power (watt). Three important aspects of the performance of the pyroelectric sensor have been studied. Firstly, measurements have revealed that the temperature dependent sensitivity increases over the range from approximately 20°C to 30°C at a rate of +1.6% °C(-1). Studies point to the key role that the properties of both the absorbing backing layer and pvdf membrane play in controlling the sensor response. Secondly, the high sensitivity of the technique has been demonstrated using an NPL Pulsed Checksource, a 3.5 MHz focused transducer delivering a nominal acoustic power level of 4 mW. Finally, proof-of-concept of a new type of acoustic sensor responding to time-averaged intensity has been demonstrated, through fabrication of an absorber-backed hydrophone of nominal active element diameter 0.4 mm. A preliminary study using such a device to resolve the spatial distribution of acoustic intensity within plane-piston and focused 3.5 MHz acoustic fields has been completed. Derived beam profiles are compared to conventional techniques that depend on deriving intensity from acoustic pressure measurements made using the sensor as a calibrated hydrophone.

Toward a reference ultrasonic cavitation vessel: Part 2--investigating the spatial variation and acoustic pressure threshold of inertial cavitation in a 25 kHz ultrasound field.

As part of an ongoing project to establish a reference facility for acoustic cavitation at the National Physical Laboratory (NPL), carefully controlled studies on a 25 kHz, 1.8 kW cylindrical vessel are described. Using a patented high-frequency acoustic emission detection method and a sonar hydrophone, results are presented of the spatial variation of inertial acoustic cavitation with increasing peak-negative pressure. Results show that at low operating levels, inertial acoustic cavitation is restricted to, and is strongly localized on, the vessel axis. At intermediate power settings, inertial acoustic cavitation also occurs close to the vessel walls, and at higher settings, a complex spatial variation is seen that is not apparent in measurements of the 25 kHz driving field alone. At selected vessel locations, a systematic investigation of the inertial cavitation threshold is described. This was carried out by making simultaneous measurements of the peak-negative pressures leading to inertial cavitation and the resultant MHz-frequency emissions, and indicates an inertial cavitation threshold of 101 kPa +/- 14% (estimated expanded uncertainty). However, an intermediate threshold at 84 kPa +/- 14% (estimated expanded uncertainty) is also seen. The results are discussed alongside theoretical predictions and recent experimental findings.

Calibration and measurement issues for therapeutic ultrasound.

This review paper examines some of the issues relating to calibration and measurement of therapeutic medical ultrasonic equipment (MUE). This is not intended to be an all-encompassing review of all aspects of characterising therapeutic ultrasound. Instead it concentrates on issues related to the acoustic output of two applications: physiotherapy and high intensity focused ultrasound surgery (HIFUS or HIFU; also referred to as high intensity therapeutic ultrasound, HITU). Physiotherapy has a well-established standards infrastructure for calibration: the requirements are small in number and well-defined. The issue for physiotherapy is not so much 'How to calibrate?' but rather, 'How to ensure that equipment IS calibrated?' The situation in the much newer area of HIFU is very different: the first steps towards writing standards are just starting and even the very basic questions of what to measure and with what type of sensor are open for debate. Readers whose main interest is in other ultrasound therapies will find ideas of relevance to their own specialty.

Acoustic emission spectra from 515 kHz cavitation in aqueous solutions containing surface-active solutes.

The effect of adding surface-active solutes to water being insonated at 515 kHz has been investigated by monitoring the acoustic emission from the solutions. At low concentrations (<3 mM), sodium dodecyl sulfate causes marked changes to the acoustic emission spectrum which can be interpreted in terms of preventing bubble coalescence and declustering of bubbles within a cavitating bubble cloud. By conducting experiments in the presence of background electrolytes and also using non-ionic surfactants, the importance of electrostatic effects has been revealed. The results provide further mechanistic evidence for the interpretation of the effect of surface-active solutes on acoustic cavitation and hence on the mechanism of sonochemistry. The work will be valuable to many researchers in allowing them to optimize reaction and process conditions in sonochemical systems.

Towards a reference ultrasonic cavitation vessel. Part 1: preliminary investigation of the acoustic field distribution in a 25 kHz cylindrical cell.

The acoustic field produced by a 25 kHz, 25 l cylindrical sonochemical processing cell has been characterised systematically using a sonar hydrophone, with the aim of establishing it as a reference test bed on which future investigations into acoustic cavitation activity may be based. Data acquired at sonication levels up to 500 W have shown that though significant cavitation activity is generated throughout the vessel, the acoustic field generated is reproducible, typically to +/- 12%. The increases in acoustic pressure are shown to be nonlinear with applied power, suggesting an intermediate optimum level for future study.

Transfer standard device to improve the traceable calibration of physiotherapy ultrasound machines.

Ultrasound (US) physiotherapy as a clinical treatment is extremely common in the Western world. Internationally, regulation to ensure safe application of US physiotherapy by regular calibration ranges from nil to mandatory. The need for a portable power standard (PPS) has been addressed within a European Community (EC)-funded project. This PPS consists of an electrical driver, a set of US transducers and a cavitation detector (CD). Each component has been extensively tested for stability and travel robustness. Transducer output power has been determined with an uncertainty of <3.3% and with a long-term (2-y) output stability of better than 3%. The CD can detect bubble activity for powers above 3 W for a 1-MHz transducer. Travel trials demonstrated the utility of the PPS in practical measurement environments. Deviations in power measurements observed during these trials were mostly acceptable (<10%), although there were also examples of gross differences (>100%). The PPS is now ready to be used to underpin traceable calibration of physiotherapy devices.

Studies of a novel sensor for assessing the spatial distribution of cavitation activity within ultrasonic cleaning vessels.

This paper describes investigations of the spatial distribution of cavitation activity generated within an ultrasonic cleaning vessel, undertaken using a novel cavitation sensor concept. The new sensor monitors high frequency acoustic emissions (>1 MHz) generated by micron-sized bubbles driven into acoustic cavitation by the applied acoustic field. Novel design features of the sensor, including its hollow, cylindrical shape, provide the sensor with spatial resolution, enabling it to associate the megahertz acoustic emissions produced by the cavitating bubbles with specific regions of space within the vessel. The performance of the new sensor has been tested using a 40 kHz ultrasonic cleaner employing four transducers and operating at a nominal electrical power of 140 W under controlled conditions. The results demonstrate the ability of the sensors to identify 'hot-spots' and 'cold-spots' in cavitation activity within the vessel, and show good qualitative agreement with an assessment of the spatial distribution of cavitation determined through erosion monitoring of thin sheets of aluminium foil. The implications of the studies for the development of reliable methods of quantifying the performance of cleaning vessels are discussed in detail.

A finite-element model of the aperture method for determining the effective radiating area of physiotherapy treatment heads.

This paper describes a theoretical study of the way in which a circular aperture placed in front of a plane-piston modifies the ultrasonic field it generates. Specifically, the radiated acoustic power transmitted by the aperture and the radiation force experienced by an absorbing target placed in the transmitted beam, are evaluated at a distance from the exit-side of the aperture. The calculations used a finite element (FE) method, in conjunction with a surface Helmholtz integral formulation to solve the fluid/structure interaction problem. The PAFEC (Program for Automatic Finite Element Computation) vibroacoustics software was used for the FE calculations and the implementation of the surface Helmholtz integral formulation method. Acoustic pressures and particle velocities were computed at required points, whilst accounting for the presence of the aperture in the medium, together with its dynamic properties when subjected to an incident sound field. This enabled the calculation of the radiation force on the absorber and its variation with the applied aperture diameter was investigated. As part of the validation process for the new FE aperture model, the ratio of radiation force to radiated acoustic power obtained using the FE method in the unapertured case, through the use of the Rayleigh integral, yielded good agreement with results obtained through an analytical solution. The study has been carried out to provide a better understanding of the factors affecting the measurement uncertainty for the aperture method of determining the effective radiating area (A(ER)) of physiotherapy ultrasound treatment heads.

Acoustic emission from cavitating solutions: implications for the mechanisms of sonochemical reactions.

The acoustic emission from collapsing cavitation bubbles generated using ultrasound of 20 kHz and 515 kHz frequencies in water has been measured and correlated with sonoluminescence and hydroxyl radical production to yield further information on the frequency dependence of sonochemical reactions. A reasonable correlation was found, and the results suggest differences in the predominant types of cavitation observed under laboratory conditions.

High-frequency acoustic emissions generated by a 20 kHz sonochemical horn processor detected using a novel broadband acoustic sensor: a preliminary study.

This paper describes the application of a novel broadband acoustic sensor to evaluating the acoustic emissions from cavitation produced by a typical commercial 20 kHz sonochemical horn processor. Investigations of the reproducibility of the processor, and of the variation in cavitation emissions as a function of output setting and sensor location are described, and resulting trends discussed in terms of the broadband integrated power in the megahertz frequency range. Companion studies with a conventional membrane hydrophone have illustrated for the first time that cavitation emissions produced by a sonochemical horn processor can extend to frequencies beyond 20 MHz, and the sensor shows that significant nonlinearity can be seen in measured cavitation activity with increasing nominal output power.

A novel sensor for monitoring acoustic cavitation. Part I: Concept, theory, and prototype development.

This paper describes a new concept for an ultrasonic cavitation sensor designed specifically for monitoring acoustic emissions generated by small microbubbles when driven by an applied acoustic field. Its novel features include a hollow, open-ended, cylindrical shape, with the sensor being a right circular cylinder of height 32 mm and external diameter 38 mm. The internal diameter of the sensor is 30 mm; its inner surface is fabricated from a 110-microm layer of piezoelectrically active film whose measurement bandwidth is sufficient to enable acoustic emissions up to and beyond 10 MHz to be monitored. When in use, the sensor is immersed within the liquid test medium and high frequency (megahertz) acoustic emissions occurring within the hollow body of the sensor are monitored. In order to shield the sensor response from events occurring outside the cylinder, the outer surface of the sensor cylinder is encapsulated within a special 4-mm thick polyurethane-based cavitation shield with acoustic properties specifically developed to be minimally perturbing to the 40 kHz applied acoustic field but attenuating to ultrasound generated at megahertz frequencies (plane-wave transmission loss > 30 dB at 1 MHz). This paper introduces the rationale behind the new sensor, describing details of its construction and the materials formulation program undertaken to develop the cavitation shield.

A novel sensor for monitoring acoustic cavitation. Part II: Prototype performance evaluation.

This paper describes a series of experimental studies to evaluate the performance of newly developed sensors for monitoring broadband acoustic emissions generated by acoustic cavitation. The prototype sensors are fabricated in the form of hollow, open-ended cylinders, whose inner surface is made from a thin film of piezoelectric polymer acting as a passive acoustic receiver of bandwidth greater than 10 MHz. A 4-mm thick coating of special acoustical absorber forms the outer surface of the sensor. The layer functions as a shield to cavitation events occurring outside the hollow sensor body, allowing megahertz acoustic emissions emanating from within the liquid contained in the sensor to be monitored. Testing of the new sensor concept has been carried out within the cavitating field provided by a commercial ultrasonic cleaning vessel operating at 40 kHz whose power output is rated at 1 kW. It is demonstrated that the prototype cavitation sensors are able to record a systematic increase in the level of the high-frequency acoustic spectrum (> 1 MHz) as electrical power to the cleaning vessel is increased. Through careful control of the experimental conditions, reproducibility of the high frequency "energy" associated with the cavitation spectrum was found to be typically +25%.